LOCUS       sequence-365608-        8049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 co-expression vector encoding PhCMV-driven SEAP coupled to 
            fluorescent reporter iRFP670 and PmPGK1-driven secreted Nluc coupled
            to fluorescent reporter mTagBFP2 that is insulated via a 5' 
            insulator sequence derived from the pGL3 plasmid 
            (PhCMV-SEAP-p2A-iRFP670-pA::A2-pA::pGL3-PmPGK1-IgkSS-Nluc-p2A-mTagBF
            P2-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8049)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 8049)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pTS2317
FEATURES             Location/Qualifiers
     source          1..8049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     620..639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(858..962)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1032..1411
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1412..1615
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1566..1586
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1660..1679
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1660..1678
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3297..3353
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3360..4289
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     primer_bind     complement(4350..4367)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4356..4580
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4726..4742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4726..4742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4765..4813
                     /note="synthetic polyadenylation signal"
     misc_feature    4827..4918
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4867..4886
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        4936..5435
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4971..4994)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5368..5387
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5488..5544
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5566..6078
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6106..6162
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6181..6888
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     protein_bind    7028..7049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7166..7183)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7337..7925)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7417..7436)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8046..8049,1..788,789..857))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
       61 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
      121 gctgcaatga taccgcggct cccacgctca ccggctccag atttatcagc aataaaccag
      181 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
      241 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
      301 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
      361 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
      421 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
      481 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
      541 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
      601 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
      661 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
      721 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
      781 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
      841 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
      901 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
      961 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccaagatctc cacgcgtggt
     1021 accctcgagt tgacattgat tattgagtag ttattaatag taatcaatta cggggtcatt
     1081 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1141 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1201 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1261 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1321 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1381 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1441 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1501 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1561 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     1621 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     1681 gacccaagct gttcgaagcg gaattcacca tgactagtct gctgctgctg ctgctgggcc
     1741 tgagactgca gctgagcctg ggcatcatcc ccgtggagga ggagaacccc gacttctgga
     1801 acagagaggc cgccgaggcc ctgggcgccg ccaagaagct gcagcccgcc cagaccgccg
     1861 ccaagaacct gatcatcttc ctgggcgacg gcatgggcgt gagcaccgtg accgccgcca
     1921 gaatcctgaa gggccagaag aaggacaagc tgggccccga gatccccctg gccatggaca
     1981 gattccccta cgtggccctg agcaagacct acaacgtgga caagcacgtg cccgacagcg
     2041 gcgccaccgc caccgcctac ctgtgcggcg tgaagggcaa cttccagacc atcggcctga
     2101 gcgccgccgc cagattcaac cagtgcaaca ccaccagagg caacgaggtg atcagcgtga
     2161 tgaacagagc caagaaggcc ggcaagagcg tgggcgtggt gaccaccacc agagtgcagc
     2221 acgccagccc cgccggcacc tacgcccaca ccgtgaacag aaactggtac agcgacgccg
     2281 acgtgcccgc cagcgccaga caggagggct gccaggacat cgccacccag ctgatcagca
     2341 acatggacat cgacgtgatc ctgggcggcg gcagaaagta catgttcaga atgggcaccc
     2401 ccgaccccga gtaccccgac gactacagcc agggcggcac cagactggac ggcaagaacc
     2461 tggtgcagga gtggctggcc aagagacagg gcgccagata cgtgtggaac agaaccgagc
     2521 tgatgcaggc cagcctggac cccagcgtga cccacctgat gggcctgttc gagcccggcg
     2581 acatgaagta cgagatccac agagacagca ccctggaccc cagcctgatg gagatgaccg
     2641 aggccgccct gagactgctg agcagaaacc ccagaggctt cttcctgttc gtggagggcg
     2701 gcagaatcga ccacggccac cacgagagca gagcctacag agccctgacc gagaccatca
     2761 tgttcgacga cgccatcgag agagccggcc agctgaccag cgaggaggac accctgagcc
     2821 tggtgaccgc cgaccacagc cacgtgttca gcttcggcgg ctaccccctg agaggcagca
     2881 gcatcttcgg cctggccccc ggcaaggcca gagacagaaa ggcctacacc gtgctgctgt
     2941 acggcaacgg ccccggctac gtgctgaagg acggcgccag acccgacgtg accgagagcg
     3001 agagcggcag ccccgagtac agacagcaga gcgccgtgcc cctggacgag gagacccacg
     3061 ccggcgagga cgtggccgtg ttcgccagag gcccccaggc ccacctggtg cacggcgtgc
     3121 aggagcagac cttcatcgcc cacgtgatgg ccttcgccgc ctgcctggag ccctacaccg
     3181 cctgcgacct ggcccccccc gccggcacca ccgacgccgc ccaccccggc tacagcagag
     3241 tgggcgccgc cggcagattc gagcagaccg ctagtggtgg ttctggtgga agcggagcta
     3301 ctaacttcag cctgctgaag caggctggtg acgtggagga gaaccctgga cctgctagtg
     3361 cgcgtaaggt cgatctcacc tcctgcgatc gcgagccgat ccacatcccc ggcagcattc
     3421 agccgtgcgg ctgcctgctt gcctgcgacg cgcaggcggt gcggatcacg cgcattacgg
     3481 aaaatgccgg cgcgttcttt ggacgcgaaa ctccgcgggt cggtgagcta ctcgccgatt
     3541 acttcggcga gaccgaagcc catgcgctgc gcaacgcact ggcgcagtcc tccgatccaa
     3601 agcgaccggc gctgatcttc ggttggcgcg acggcctgac cggccgcacc ttcgacatct
     3661 cactgcatcg ccatgacggt acatcgatca tcgagttcga gcctgcggcg gccgaacagg
     3721 ccgacaatcc gctgcggctg acgcggcaga tcatcgcgcg caccaaagaa ctgaagtcgc
     3781 tcgaagagat ggccgcacgg gtgccgcgct atctgcaggc gatgctcggc tatcaccgcg
     3841 tgatgttgta ccgcttcgcg gacgacggct ccgggatggt gatcggcgag gcgaagcgca
     3901 gcgacctcga gagctttctc ggtcagcact ttccggcgtc gctggtcccg cagcaggcgc
     3961 ggctactgta cttgaagaac gcgatccgcg tggtctcgga ttcgcgcggc atcagcagcc
     4021 ggatcgtgcc cgagcacgac gcctccggcg ccgcgctcga tctgtcgttc gcgcacctgc
     4081 gcagcatctc gccctgccat ctcgaatttc tgcggaacat gggcgtcagc gcctcgatgt
     4141 cgctgtcgat catcattgac ggcacgctat ggggattgat catctgtcat cattacgagc
     4201 cgcgtgccgt gccgatggcg cagcgcgtcg cggccgaaat gttcgccgac ttcttatcgc
     4261 tgcacttcac cgccgcccac caccaacgcg ctagcggatc caccggtgtc tagaaagctt
     4321 tgaggccggc ctgcaggccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     4381 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4441 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4501 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     4561 ggatgcggtg ggctctatgg tagcttagga ggcggaaaga accagcgcgg gagaccgtca
     4621 gttggtctcc agctatacag gtgcaccgct ttataataaa actgatacag agaacgttat
     4681 catgtgtgtt ggtttttttt gtgtgtgcgg tttaagggag cctcggtcat agctgtttcc
     4741 tgtgtaaaac gacggccagc gcgtaataaa atatctttat tttcattaca tctgtgtgtt
     4801 ggttttttgt gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa
     4861 aacaaactag caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctgg
     4921 taccctcgag ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     4981 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     5041 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     5101 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     5161 aaatggaagt agcacgtctc agtagtctcg tgcagatgga cagcaccgct gagcaatgga
     5221 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     5281 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     5341 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     5401 cgctgttctc ctcttcctca tctccgggcc tttcgacctc cagcccgagc tcggtatagg
     5461 ttcgaagcgg aattcaccat gactagtgag acagacacac tcctgctatg ggtactgctg
     5521 ctctgggttc caggttccac tggtgacgct agtggtggtt ctggtatggt cttcacactc
     5581 gaagatttcg ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa
     5641 cagggaggtg tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg
     5701 attgtcctga gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa
     5761 ggtctgagcg gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg
     5821 gatgatcatc actttaaggt gatcctgcac tatggcacac tggtaatcga cggggttacg
     5881 ccgaacatga tcgactattt cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa
     5941 aagatcactg taacagggac cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc
     6001 aaccccgacg gctccctgct gttccgagta accatcaacg gagtgaccgg ctggcggctg
     6061 tgcgaacgca ttctggcggc tagtggtggt tctggtggaa gcggagctac taacttcagc
     6121 ctgctgaagc aggctggtga cgtggaggag aaccctggac ctgctagtgg tggttctggt
     6181 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
     6241 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     6301 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     6361 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     6421 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     6481 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     6541 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     6601 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     6661 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca
     6721 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     6781 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     6841 gtggccagat actgcgacct ccctagcaaa ctggggcaca aacttaatgc tagcggatcc
     6901 accggtgtct agaaagctca gaaattatga gagcgtttaa taaagtattg actatggttg
     6961 acaaaacgtg tgttggagtt ttttgtgtgc tttgtcatca actgagccag taggtcgagg
     7021 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     7081 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7141 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     7201 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     7261 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     7321 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7381 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     7441 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7501 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     7561 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     7621 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7681 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7741 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     7801 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7861 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7921 ctcaagaaga tcctttgatc ttttctacgg ggattatcaa aaaggatctt cacctagatc
     7981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     8041 gacagttac
//
